PROJECT SUMMARY The ventricular conduction system (VCS) is critically important for normal myocardial excitation and contraction. Heritable and acquired syndromes perturbing VCS formation or function are responsible for a substantial burden of cardiovascular disease, including heart block, triggered and reentrant arrhythmias, sudden cardiac death, as well as myocardial dyssynchrony and progression of heart failure. Using genetically encoded conduction system reporter mice, comparative transcriptional profiling, and a battery of functional assays, our laboratory has pioneered studies of VCS development, physiology and disease pathogenesis. Through this approach, we have identified and characterized the transcriptional signature of Purkinje cells and identified a number of novel genes that regulate diverse aspects of VCS biology, including transcription factors, ion channel subunits and regulatory proteins. In the current application, we propose to establish a more global and comprehensive understanding of gene regulatory networks that are operative in this rare population of excitable cells, by extending our analysis to include microRNAs that are differentially expressed in the VCS. More specifically, we propose to: determine the cell autonomous requirements for microRNAs in the VCS; identify microRNAs required for Purkinje cell differentiation using functional genomics screens; and define Purkinje cell transcriptional signatures and electrophysiology in health and disease.